As iron (Fe)-related disorders are prevalent world-wide, understanding Fe homeostasis is critical to understanding human health. Fe homeostasis is largely regulated by hepcidin, a liver-derived peptide that inhibits dietary Fe absorption and macrophage Fe recycling. Hepcidin expression is inhibited and stimulated respectively by increased erythropoietic drive and Fe overload. Focused on identifying soluble regulators of hepcidin expression, my current work has implicated transferrin (TF), the abundant serum metal-binding protein, and manganese (Mn), an essential TF-bound metal, in the regulation of hepcidin expression. My first objective, to be completed during the mentored phase, is to determine the effect of TF on hepcidin expression in vivo. I will construct transgenic mice expressing TF variants with altered metal affinity, breed them onto a TF-deficient background and evaluate hepcidin expression before and after bone marrow ablation to minimize the inhibition of hepcidin expression by erythropoietic drive. The second objective, to be initiated during the mentored phase, is to identify commonalities and divergences between cellular Mn and Fe homeostasis as a means to further dissect Fe homeostasis. I will assay 54Mn and 55Fe transport in cell lines with altered levels of known metal transporters and determine the effect of altered Mn and Fe levels on 54Mn and 55Fe transport and incorporation into TF. The third objective, the focus of the independent phase, is to identify novel factors required for cellular metal homeostasis. As high Mn levels are toxic to many cell lines, I will alter cellular gene expression with shRNA and cDNA libraries, select for cells resistant to high Mn levels and identify mutant lines of interest by 54Mn and 55Fe transport assay. From the first two objectives, I will achieve proficiency in the study of Fe transport and distribution;from the last two objectives, I will establish an independent focus divergent from my mentor's, the role of iron in hematologic disorders. This proposal reflects the overall trend in my research-the study of mammalian metal homeostasis and its relation to human health and disease-and will lead to a paradigm of metal homeostasis from which novel treatments for metal-related disorders can be designed. PUBLIC HEALTH RELEVANCE: The goal of this study is to determine how iron in the body stimulates the liver to produce a hormone that limits iron absorption from the gut. The results of this research should offer novel treatment options for iron-related disorders such as anemia.